Enteric bacilli differential apparatus

ABSTRACT

THIS ENTERIC DIFFERENTIAL APPARATUS COMBINES FOURTEEN RECOGNIZED BIOCHEMICAL PARAMETERS IN A FOUR-TUBE CULTURE MEDIA IN SUCH A MANNER THAT EACH INDIVIDUAL SPECIES WITHIN THE FAMILY ENTEROBACTERIACEAE CAUSES THE MEDIA TO ASSUME A DISTINCTIVE APPEARANCE. TWO TUBES COMPRISE THE BASIC R/B DIFFERENTIAL SYSTEM. TWO ADDITIONAL TUBES COMPRISE AN EXPANDER SET. ONE OR BOTH OF THE EXPANDER TUBES MAY BE USED IN COMBINATION WITH THE BASIC SYSTEM. A UNIQUE TUBE FACILITATES INOCULATION, SEPARATES CULTURE MEDIA COMPONENTS TO PREVENT INTERACTION, THUS INCREASING SHELT LIFE, PROVIDES SHARP DIFFERENTIATION BETWEEN REACTIONS AND PERMITS THE USE OF SEMI-SOLID GELS TO OBTAIN MORE SENSITIVE DETERMINATION OF MOTILITY. THE TUBE MAY BE USED FOR OTHER CULTURE MEDIA TESTS. ANOTHER ADVANTAGE IS THAT THE TUBE PERMITS TRANSPORTATION OF SEMI-SOLID GEL MEDIA WITHOUT BREAKUP.

Aug. 20, 197% Q, A. BECKFD 3,830,703

ENTERIC BACILLI DIFFERENTIAL APPARATUS Filed Feb. 2, 1973 k 3 Sheets-Sheet 1 LOCATION OF REACTIONS THE EXPANDERS 7 BASIC SYSTEM Positive CIT/RHAM SORANASE d Pos. or Neg.

Negative Delayed Positive Reaction E Citrate B Note Appeorunce of Roffinose 3O visible growth on the slant should be Indole interpreted as citrate positive. i

v, V m Orn r hme Rhomnose Arobinose Motility Q. A. BECKFRD ENTEHIC BACILLI DIFFERENTIAL APPARATUS Filed Feb. 2. 1973 GREEN PROT EUS VU LGARIS 3 Sheets-Sheet 2 FIGS READ PHENYLALANINE TEST Brown Slant Denotes PosItive Phenylalanine Deaminase this is the PROTEUS-PROVIDENCIA Group PROTEUS MlRABlLlS PROTEUS MORGAN PROTEU'S RETTGERI H23 H 3 OR PROVlDENCIA Ornithine Ornithine -l OrniIhine lndole lndole Ornithine W additional test required NON HES PRODUCING RE E P, MIRABlLlS F3 Refige" Providencia STEP 2 EDWARDSIELLA Lysine lndole If Slant is Purple or Yellow (Negative Phenylalanine) READ H 8 TEST Black Upper Butt Denotes Positive H 8 SALM ONE LL A-ARIZONA Lysine lndole additional test required 1. DULClTOL/MALONATE Salmonella Arizona 2.SEROTYPE SALMONELLA-TYPHI Lysine Ornithine lndole No gas produced the EDWARDSlELLA-SALMONELLA ARIZONA- CiTROBACTER Group CITROBACTER Lysine NOTE: LACTOSE POSITIVE AND NEG- ATlVE TUBES PICTUREDO SMALL TUBE REACTIONS ARE NON-CONTRIB- UTORY TO DIFFERENTIATION.

Filed Feb. 2, 1.973

Gas produced Motility d Q. A BECKF@D ENTERIC BACILLI DIFFERERTIAL APPARATUS STEP 3 If Slant is Purple or Yellow and H 8 is Negative Pink Swab Denotes Positive indole this is the E. COLl-SHIGELLA Group Ecoli may be positive or negative in its ornithine. lysine, lactose and motility reactions.

Note; 60% Shigell a are indole negative. Colony morphology, negative lysine, negative motility, lack of gas, will indicate ShigeIIa. (Serotype) Some Klebsiella are indole positive.

E. COLI Gas produced Motility NNEl Ornithine Motility Lysine No gas produced 5 sheete Sheet 5 READ lNDOLE TEST: FIGG Ornithine Motility Lysine No gas produced Certain biotypes of S. flexneri 6 produce gas.

additional tests required indole positive Klebsiella.

Yellow Swab Denotes Negative indole this is the KLEBSIELLA-ENTEROBACTER-SERRATIA Group A few Klebsiello will be indole positive. In such casestheir colony morphology, coupled with their r/b reactions (i.e. non-motile, negative ornithine), will indicate the organism to be an ENTEROBACTIgR Ornithine Orn thine Motility Motility Lysine Abundant gas lndole d Lactose G05 Produced additional test, Rhamnose RHAMNOSE (r/b)LYSlNE E. cloacae E. HAFNIA or non H28 TYP ICAL SERRATIA United States Patent 01 ice 3,830,703 Patented Aug. 20, 1974 Int. Cl. C12k N US. Cl. 195-127 6 9 Claims ABSTRACT OF THE DISCLOSURE This enteric differential apparatus combines fourteen recognized biochemical parameters in a four-tube culture media in such a manner that each individual species within the family Enterob-acteriaceae causes the media to assume a distinctive appearance. Two tubes comprise the basic R/B differential system. Two additional tubes comprise an expander set. One or both of the expander tubes may be used in combination with the basic system. A unique tube facilitates inoculation, separates culture media components to prevent inter-action, thus increasing shelf life, provides sharp differentiation between reactions and permits the use of semi-solid gels to obtain more sensitive determination of motility. The tube may be used for other culture media tests. Another advantage is that the tube permits transportation of semi-solid gel media without breakup.

The aforementioned Abstract is neither intended to define the invention of the application which, of course, is measured by the claims, nor is it intended to be limiting as to the scope of the invention in any way.

BACKGROUND OF THE INVENTION For many years microbiologists throughout the world have been identifying Enterbacteriaceae by a combination of colony morphology, biochemical reactions and serology. The biochemical aspects of the identification creates the greatest burden in work application in the clinical laboratory. Several tubes of media are required for proper biochemical determination and as many as eighteen individual tubes are recommended by the governmental agencies. To overcome this task and alleviate the tremendous number of tubes required, clinical laboratories have designed their own initial secreening system using four to six tubes. The ones most commonly encountered are TSI, Kliglers or iRussels double sugar, Simmons Citrate, M-R-VP, K'CN, Urea, Phenylalanine, Malonate, SIM, Lactose, Glucose and Maltose.

The initial screening most often requires many additional biochemical tests and the choice and judgment used depending on the level of the technology requires 72 hours or more for proper identification of the organism.

In the copending application of Rollender and Beckford. Ser. No. 14,805, filed Mar. 2, 1970, there was disclosed a system known commercially as the R/B (-Rol lender/Beckford) system. This prior system standardizes the initial screening system by establishing a constant and reduced the number of tubes required to two. It was established that biochemical reactions in combination could compare favorably with individual biochemical determinations. However, it was found that in many cases it was desirable to be able to simultaneously speciate Within the groups and detect the rarer atypical organisms. Accordingly, two tubes were added to the system which achieve this goal.

The prior art apparatus was subject to limited shelf life and it was found that diffusion problems existed, which created ambiguous readings. The present application discloses a tube designed to eliminate these problems.

SUMMARY OF THE INVENTION The present system employs constricted tubes. The constriction is about 1 inch from the bottom of the tubes, 2 mm. in internal diameter and 4 mm. in length. Preferably, the constriction is tapered and in cross-section resembles a pair of opposed arcs. The two mm. internal diameter permits the entrapment of an Air Bubble. This physical separation of the two media, arrested diffusion, prolonged shelf life and resulted in cleaner sharper color reactions. The test media contained in three or four such tubes is described more fully hereinafter.

DESCRIPTION OF THE DRAWING FIG. 1 is a perspective view of the media tube of this invention;

FIG. 2 is a sectional view taken along lines 22 of FIG. 1;

FIG. 3 is a schematic showing of a set of tubes containing diagnostic media with legends indicating areas where particular results are to be observed;

FIG. 4 is a schematic showing of a set of tubes containing diagnostic media with legends indicating areas where particular results are to be observed;

FIG. 5 is a schematic showing of steps 1 and 2 of .a test for differentiating enteric bacteria using the tubes of FIG. 4; and

FIG. 6 is a schematic showing of step 3 of the test.

DESCRIPTION OF PREFERRED EMBODIMENT A tube 10 made of transparent glass or synthetic resin is used to contain the test media. The tube is distinguished by a constricted connecting passage 12 about 4 mm. long and about 2 mm. wide. Preferably the constriction is in the form of opposed arcs as viewed in cross-section. The lower portion 14 is used for one media formulation 15 while the upper portion 16 is used for a different formulation 17 The passage 12 may be filled with a sterile gas or fluid such as oil to prevent interaction between formulations 15, 17. A screw cap 18 is provided which mates with threads 20. In a particular test system described hereinafter a set of four tubes is employed, tubes B, C and D being slightly smaller than tube A.

Description of the tubes Tube A: The lower butt portion of the tube is filled with a gel containing lysine and means for the detection of the decarboxylation of the lysine in the presence of bacilli causing the decarboxylation of the lysine.

The upper butt portion is then filled with a gel, nontoxic to bacteria, for which the test is to be made and having dispersed in the gel the following:

(1) sufficient nutrient to support the growth of bacilli to be differentiated;

(2) sufficient amino acid and a sufiicient source of available iron to provide an indication of deamination of the amino acid by selected bacilli. The iron may be supplied in the form of ferrous sulphate, ferris ammonium citrate or combinations thereof;

(3) sufiicient carbohydrate to provide an indication of fermentation and to act as a catalyst for decarboxylation;

(4) sufficient electrolyte to permit the flow of ions through the gel;

(5) a source of sulphur for enhancing production of H 5 in the presence of selected bacilli; and

(6) apH indicator.

Tube B: In the smaller tube there is provided means for the detection of indole produced by select-ed bacilli as well as orinthine decarboxylation and motility. In the past it 'was necessary to utilize a high concentration of agar in the gel to provide sufiicient rigidity to prevent break-up during shipment. The rigid gel is relatively insensitive to motility. By reducing the percentage of agar present from the prior 0.4% to 0.3%, a much more reliable and easily read motility test is provided.

Motility in the tube B is read as positive when the appearance is cloudy making the stab line barely visible. It is also positive when, though not particularly cloudy, there is evidence of growth progressing in all directions completely around the stab line.

Motility is read as negative when the appearance is clear making the stab line produced by the inoculation wire clearly visible. It is also negative when there is appearance of growth in one or two directions only from the stab line which is due to gas production, causing a hairline split in the media which introduces growth of organisms in the split.

The indole test is carried out by wetting a swab 20 in indole reagent and then touching the wetted swab to the media surface of the tube B. If positive, the swab will turn pink; if negative, the swab remains yellow. This is shown in FIG. 6.

It will be noted from FIG. 5 that the reaction for Proteus rettgeri and Providencia are similar and accordingly and additional test is required to distinguish between them. This further test is carried out in Tube C. Typically sixty-eight percent of Proteus rettgeri will provide a positive reaction in lower portion of Tube C which contains Rhamnose. Accordingly, a positive reaction is a definitive indication. If, however, a negative reaction results, a further test is made employing Urease to distinguish between P. rettgeri and Providencia. The urease test is a standard test which does not form part of this invention. Thus, by inoculating Tube C at the same time as Tubes A and B, a day of testing may be saved.

To further difierentiate the Enterobacter-Serratia Group, which includes those bacteria characterized by negative lactose, positive lysine, negative indole response and positive ornithine response, tube D is employed. It should be noted that H 8 negative Salmonella masquerades in this group.

The reactions taking place in tubes C and D are shown in FIG. 4.

General Test Procedure To inoculate the media, a four-inch inoculation wire with a flattened loop (needle eye) terminus is flame sterilized, allowed to air cool, and then touched to the center of a selected, well isolated colony growing on an appropriate primary plating media. The needle is stabbed into the bottom of the tube. The tapered portion 22 serves to guide the needle into the lower butt portion 14. An important advantage of the guide is that the inoculation always takes place in the center of the media mass providing uniformity between tests.

As the needle is withdrawn, it is gently streaked with a back-and-forth movement across the slant surface 24. The needle is then jabbed into the butt of a smaller tube 10.

The tubes are then incubated at 37 C. for 18 to 24 hours with caps 18 loosely positioned to allow air to enter for the aerobic reactions to take place.

Detailed Procedure I. A sample to be diagnosed, such as feces or urine, is swabbed on to an enteric isolation medium, i.e., Mac- Conkey, EMB, XLD. The plates are streaked starting in the swabbed area and moving out into the uninoculated portions of the plate in a pattern that creates a dilution of the sample and promotes growth of tiny isolated colonies of bacteria B B etc.

II. The inoculated plates are then incubated at 37 C. for a period of 24 hours.

III. A 4" flattened loop is flamed and allowed to air cool and then touched to the center of a well-isolated colony on the incubated plate. The standard oxidase test is performed. If a negative result is obtained, the

4 organism is tested for the differentiation of Enterobacteriaceae.

IV. The large tube is inoculated to the bottom and the slant streaked lightly on withdrawal of the loop. Without reflaming loop, the small tube B is inoculated by stabbing to the bottom leaving a clean stab line. Tubes C and D are then inoculated without reflaming in the same manner as Tube A.

V. The inoculated tubes are then incubated at 37 C. for 18 and not more than 24 hours with loose caps.

VI. The tubes are then read.

If a brown slant appears, a positive phenylalanine is indicated showing the presence of the Proteus-Providencia Group. Species within this group can be identified by the following:

Proteus vulgaris:

H 5 Ornithine Proteus mirabilis:

H 8 Ornithine Indole Proteus morganii:

H S Ornithine Indole Norm: Check indole for non HzS producing P. mirabilis.

In FIG. 3, there is shown the location of the reactions in tubes A and B. The slant portion of the A tube upper section contains phenylalanine and lactose, the lower portion of the upper section provides glucose and H S while the lower chamber 14 contains a lysine test. The B tube upper chamber contains indole and the lower chamber 14 an ornithine motility test.

In FIG. 4 there is shown the locations of the reactions in tubes C and D.

In FIGS. 5 and 6 the citrate reaction is shown within the triangles, while the tests of tube D are shown at the bottom of FIG. 6. The latter tests may be summarized as follows:

Hrs negative E. llq- Saluefa- Ser- E. hafm0- ciem ratia m'ue nella d T l(+) i d t t Norn.d or Positive reaction. Negative reaction Delayed positive reaction.

Tube A-Butt Portion Peptone 15.0 partsiLO by weight. Yeast extract 3.0 partsi2.0 by weight.

. Phenylalanine 3.0 parts-$0.5 by weight. Tryptophan 2.0 partsi0.5 by weight. Dextrose 1.0 partsi0.5 by weight. Lactose 10.0 partsiLO by weight. Sodium chloride 5.0 partsiLO by weight. Ferric ammonium citrate 0.5 parts-$0.5 by weight. Sodium thiosulfate 0.5 partsilLS by weight. Agar 15.0 partsiLO by weight. Brom Cresol-Purple 0.02 partsi0.001 by weight.

Water 1000 parts by weight.

5 Tube A-Slant Portion Peptone 5.0 partsiLO by weight. Y t xtract 3.0 parts-$2.0 by weight, Lysine 10.0 parts:0.8 by weight. Dextrose 1.0 partsi0.5 by weight. g 6.0 partsi0.3 by weight. Brom Cresol-Purple 0.02 partsi0.00l by weight. Water 1000 parts by weight.

Tube B Peptone 20.0 partsiLO by weight.

Yeast extract 3.0 partsizo by Weight.

L-ornithine 5.0 parts:1.0 by weight.

Tryptophan 5.0 partsiLO by weight.

Dextrose 1.0 parts-$0.5 by weight.

Agar 15.0 partsiOj by weight.

Brom Cresol-Purple 0.02 parts:0.001 by weight.

Water 1000 parts by welght.

Range, gins.

water,

Tube C-Citrose/rhamnose High preferred Low Butt:

Tryptona. 10.0 3.0 0.5 Sodium chloridfinnilut 07 12 (i fi fflfiifi flfju 112 15. 25. B th ol blue. 1. 0.

R lfa mno i 15. 0 5. 0 2. 0 pH 7. 8 7. 1 6.

Slant:

Ammonium dihydrogen phosphate 3. 0 1. 0 0. 5 Dipotassium phosphate 3.0 1.0 0.5 Sodium chloride 8. 0 5. 0 1. 0 Sodium eitrate 3. 0 2. 0 0. 5 Magnesium sulfate- 0. 5 0.2 0. 09 A as at at h lbl g iiyr nf? 7.4 6.9 6.4

Range, gms./l water, Tube D-Soranese High preferred Low 10.0 2.0 0.5 Sodium chloride 8. 0 5. 0 1. 0 Dipotassium phsophat 1. 0 0. 6 0. 1 Agar 17. 0 14. 5 2. 5 Brom Tyhmol Blue- 0. 08 0. 03 0. 01 Brom Creso1Purple-- 0. 015 0.01 0. 005 Arabinose 10. 0O 3. 0 0. 5 pH l 1 6- 8.0 7.4 6.7

Slant (u perc am er:

Des xyribonuleic acid 15:8 (2) 8. 0 5. 0 1. 0

There has been disclosed heretofore the best emodimerit of the invention presently contemplated. However, it is to be understood that various changes and modifications may be made by those skilled in the art without departing from the spirit of the invention.

What I claim is:

1. An enteric bacilli difierentiation apparatus comprising a plurality of transparent tubes having upper and lower chambers joined by a conduit of reduced crosssectional area containing media in the upper and lower chambers separated by the conduit for making determina- (c) another of said tubes contains media for citrate determination in the upper chamber and media for rhamose determination in the lower chamber.

2. The apparatus of Claim 1 including a fourth said tube containing media in the upper chamber for DNASE, Ratfinose and Sorbital determinations and media in the lower chamber for Arabinose determinations.

3. The apparatus of Claim 1 wherein said conduits contain a gas separating the contents of the respective upper and lower chambers.

4. An enteric bacilli differentiation apparatus containing media in combination comprising;

(I) a first transparent container comprising a first chamber and a second chamber joined by a conduit of reduced cross-sectional area containing a first media in said first chamber and a second media in said second chamber spaced from said first media by said conduit;

(a) said first media comprising a gel non-toxic to the bacilli to be differentiated having dispersed therein:

(1) sutficient nutrient to support the growth of bacilli to be diiferentiated;

(2) sufficient amino acid and a sufiicient source of ferric ions to provide an indication of deamination of the amino acid by selected bacilli;

(3) sufiicient carbohydrate to provide an indi cation of fermentation and to act as a catalyst for decarboxylation;

(4) a source of sulphur for enhancing production H 8 in the presence of selected bacilli; and

(5) Brom Cresol-Purple; and

(b) said second media comprising a gel containing lysine and including means for the detection of the decarboxylation of the lysine in the presence of bacilli causing the decarboxylation of the lysine;

(II) a second transparent container having a gelled media therein comprising:

(a) a nutrient for the bacilli to be differentiated;

(b) sufi'icient L-Ornithine to provide an indication of decarboxylation by selected bacilli;

(c) sufficient tryptophane to provide indole upon degradation by selected bacilli; and

(d) carbohydrate; and

(III) a transparent container having an upper and a 5. The apparatus of Claim 4 comprising in combination a further transparent container having an upper and a lower chamber connected by a conduit of reduced crosssectional area;

(a) said lower chamber containing:

Gms./l. Water Peptone 0.5-10.0 Sodium chloride 1.0-8.0 Dipotassium phosphate 0.1-1.0 5 Agar 2.5-17.0 Brom Thymol Blue 0.1-0.08 Brom Cresol Purple 0.0050.015 Arabinose 0.5-u00 pH 6.7-8.0. 10 and (b) said upper chamber containing:

Desoxyribonuleic acid 0.2-5.0 Peptone 3.0-30.0 Sodium chloride 1.0-8.0 Agar 2.5-18.0 Rafiinase 4.020.0

Sorbital 0.'1-5.0 Brom Cresol Purple 0.02-0.1 Phenol Red 0003-002 Toludine Blue 0 0.01-0.09 pH 7.4-8.0.

6. The apparatus of Claim 5 wherein said conduits contain a gas separating the contents of the respective upper and lower chambers.

7. An enteric bacilli differentiation apparatus comprising:

(-I) A first constricted transparent tube having upper and lower chambers joined by a conduit of smaller cross-sectional area, its upper portion containing a first medium and in its lower portion a second medium;

(a) said first medium comprising a gel non-toxic to the bacilli to be differentiated having dispersed therein;

(1) sutficient nutrient to support the growth of bacilli to be differentiated;

(2) sufficient amino acid and a sufiicient source of ferric ions to provide an indication of deamination of the amino acid by selected bacilli;

(3) sufficient carbohydrate to provide an indication of fermentation and to act as a catalyst for decarboxylation;

(4) a source of sulphur for enhancing production of H 5 in the presence of selected bacilli; and

(5) Brom Cresol-Purple; and

(b) said second medium comprising a gel containing lysine and including means for the detection of the decarboxylation of the lysine in the presence of bacilli causing the decarboxylation of the lysine; and

(II) A second constricted transparent tube having upper and lower chambers joined by a conduit of reduced cross-sectional area containing:

(a) In the upper and lower chambers, 21 solid medium for simultaneous indole decarboxylation reactions of amino acids and motility comprismg:

(1) nutrient;

(2) a solidifying agent;

(3) Brom Cresol-Purple;

(4), A material capable .of being fermented and producing an acid in the presence of enteric bacteria; and

(5) at least two different amino acids, namely Ornithine and Tryptophan. (III) A third constricted transparent tube having upper and lower chambers joined by a conduit of reduced cross-sectional area; said upper chamber containing Gms./l. water Ammonium dihydrogen phosphate 0.5-3

Dipotassium phosphate 0.5-3 Sodium chloride l.8. Sodium citrate 0.5-3 Magnesium sulfate 0.090.5 Agar 2.5 17.0 Brom Thymol Blue 0.01-1.0 pH 6.4-7.4.

said lower chamber containing Tryptone 0.510.0 Sodium Chloride 1.0-8.0 Dipotassium Phosphate 0.1-1.0 Agar 2.5-17.0' Brom Thymol Blue 2.1-15.0 pH 6.5-7.8.

8. The apparatus of Claim 7 wherein said conduits contain a gas separating the contents of the respective upper and lower chambers.

9. The apparatus of Claim 7 comprising in combination a further transparent container having an upper and a lower chamber connected by a conduit of reduced crosssectional area;

(a) said lower chamber containing:

' Gms./l. water Peptone 0.5-10.0

Sodium chloride 1.0-8.0 Dipotassium phosphate 0.1-1.0 Agar 2.5-17.0 Brom Thymol Blue 0.1-0.08 Brom Cresol Purple 0.005-0.015 Arabinose 0.5-10.00 pH 6.7-8.0.

and

(b) said upper chamber containing:

Desoxyribonuleic acid 1.2-5.0 Peptone 3.0-30.0 Sodium chloride a- 1.0-8.0 Agar 2.5-18.0 Rafiinase 4.020.0 Sorbital 0.1-5.0 Brom Cresol Purple 0.02-0.1 Phenol Red 0.003-0.02 Toludine Blue 0 0.01-0.09 pH 7.4-8.0.

References Cited Auxotab Catalog, Colab Laboratories Inc., 1971.

A. LOUIS MONACELL, Primary Examiner R. J. WARDEN, Assistant Examiner U.S. Cl. X.R. 

